PROJECT SUMMARY People that experience adversity during early life exhibit increased risk for a number of health disorders throughout the lifespan. For example, adverse childhood experiences predict elevated susceptibility to anxiety disorders in adulthood, increased stress reactivity, and altered immune function. However, the physiological and molecular mechanisms that connect early life experiences to health outcomes later in life remain poorly understood. Further, not everyone experiences the same consequences, suggesting that later life environment or genetic variation may exacerbate, or protect against, the negative consequences of early life insults. The biological embedding of early life experiences is thought to arise, at least in part, from persistent changes to the epigenome in response to early life conditions. DNA methylation (the addition of a methyl group to cytosine bases), has received particular attention in this regard, because DNA methylation marks can be highly stable and can influence gene regulation. In support of this idea, DNA methylation levels at thousands of genomic regions have now been associated with early life adversity. However, if these changes explain variation in human health, differential methylation at these sites must also have functional consequences for gene regulation?an assumption that is rarely empirically tested. Characterizing the subset of cases in which early adversity-associated DNA methylation variation can influence downstream traits is therefore a central priority for understanding the role of epigenetics in the health consequences of early life adversity. To address this priority, the proposed study will perform the first comprehensive test of the functional consequences of early life adversity-associated DNA methylation variation for gene regulation. Specifically, it will use a novel, high-throughput method (mSTARR-seq) to test whether DNA methylation marks alter gene regulatory activity at 10,000 candidate regions in the human genome. Because DNA methylation may be differentially important across cell types, this relationship will be investigated in three cell types with known links to the biology of early life adversity: immune cells in the blood, neural cells, and adrenocortical cells that are important in the stress response. In addition, because the effects of early adversity-associated DNA methylation may depend on genotype or other environmental stressors, we will test for methylation-dependent regulatory activity across genetically variable humans and under environmental challenges. Finally, we will use CRISPR-dCas9 epigenome editing to investigate whether precisely altered DNA methylation levels change gene regulation of individual genes. At its conclusion, the proposed work will generate the first systematic assessment of the regulatory consequences of early adversity-associated DNA methylation. Its findings will therefore provide much-needed insight into the key targets of early life adversity in the epigenome, with the goal of identifying new opportunities for treatment and intervention.